EP3135759B1 - Putrescine-producing variant strain and putrescine production method using same - Google Patents

Putrescine-producing variant strain and putrescine production method using same Download PDF

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EP3135759B1
EP3135759B1 EP15783187.6A EP15783187A EP3135759B1 EP 3135759 B1 EP3135759 B1 EP 3135759B1 EP 15783187 A EP15783187 A EP 15783187A EP 3135759 B1 EP3135759 B1 EP 3135759B1
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putrescine
microorganism
ncgl2523
corynebacterium
sequence
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French (fr)
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EP3135759A4 (en
EP3135759A1 (en
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Kyoung Min Lee
Hongxian LI
Su Jin Park
Young Lyeol Yang
Hye Won Um
Hee Kyoung Jung
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CJ CheilJedang Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01017—Ornithine decarboxylase (4.1.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the present invention relates to a recombinant microorganism for producing putrescine and a method of producing putrescine using the same.
  • Biogenic amines are nitrogen compounds mainly produced by decarboxylation of amino acids, amination of aldehyde and ketone, and transamination.
  • Such biogenic amines are low molecular weight compounds, which are synthesized during metabolism of microorganisms, plants, and animals, and thus are known as components easily found in cells thereof.
  • polyamine such as spermidine, spermine, putrescine (or 1,4-butanediamine), cadaverine, etc., are substances present in most living cells.
  • putrescine is an important raw material which synthesizes polyamine nylon-4,6 by reacting with adipic acid, and is produced mainly by chemical synthesis through acrylonitrile and succinonitrile from propylene.
  • membrane proteins encoding NCgl2522 function as a putrescine exporter in a microorganism of genus Corynebacterium , which includes a putrescine synthesis pathway, and confirmed that putrescine may be produced at a high yield by enhancing NCgl2522 activity from to the endogenous activity thereof ( KR Patent Application No. 10-2013-0030020 )
  • NCgl2523 gene is a multidrug-resistance-related transcription factor which belongs to TetR family, and is known to act as an NCgl2522 expression inhibitor ( Hirochi et. al. J Biol. Chem. 280:46, 38711-38719. 2005 ).
  • the present inventors have continuously conducted research and confirmed enhanced putrescine production by depletion of NCgl2523 which constitutes NCgl2522 operon, in the manner similar to the effects of enhancing NCgl2522 activity, thereby completing the present invention.
  • An objective of the present invention is to provide a recombinant microorganism capable of producing putrescine at high yield.
  • Another objective of the present invention is to provide a method of producing putrescine at high yield using the microorganism.
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein an activity of ornithine decarboxylase (ODC) is further introduced into the microorganism.
  • ODC ornithine decarboxylase
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which the ODC has an amino acid sequence represented by SEQ ID NO: 10.
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein acetyltransferase activity is further inactivated in the microorganism.
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein the acetyltransferase comprises an amino acid represented by SEQ ID NO: 15 or 16.
  • the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which the microorganism is Corynebacterium glutamicum.
  • the present invention provides a method of producing putrescine including:
  • the present invention provides a method of producing putrescine, in which the microorganism of genus Corynebacterium is Corynebacterium glutamicum.
  • the present invention relates to a microorganism of genus Corynebacterium having putrescine productivity, wherein a protein having an amino acid sequence represented by SEQ ID NO: 2, thus NCgl2523, is inactivated
  • NCgl2523 refers to a multidrug-resistance-related transcription factor which belongs to TetR family, and is known to act as an Ncgl2522 expression inhibitor ( Hirochi et. al., J Biol. Chem. 280(46): 38711-38719, 2005 ).
  • NCgl2523 is a protein having an amino acid of SEQ ID NO: 2 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more, much more specifically 98% or more, and most specifically 99% or more homology to the sequence, and is not limited thereto as long as it is a protein having the activity of an NCgl2522 expression inhibitor.
  • amino acid sequences of proteins showing the activity may differ depending on species or strain of microorganisms, it is not limited thereto. It is obvious that a protein having an amino acid sequence in which the sequence is partially deleted, modified, substituted, or inserted is included in the scope of the present invention, as long as a sequence having homology to the sequence shows biological activity practically equivalent or corresponding to a protein of SEQ ID NO: 2.
  • homology refers to similarity between given amino acid sequences or nucleotide sequences and may be represented in percentage.
  • the homology sequence which have identical or similar activity with a given amino acid sequence or nucleotide sequence is indicated by "% homology".
  • homology may be examined by using conventional software calculating mediated parameters such as score, identity, similarity, etc., and more specifically using BLAST 2.0, or by comparing sequences via Southern hybridization under defined stringent conditions. Appropriate hybridization conditions may be defined by the scope of the art, and may be determined by one of ordinary skill in the art using known methods (i.e., J.
  • a polynucleotide encoding NCgl2523 of the present invention may include an amino acid sequence of SEQ ID NO: 2, or a polynucleotide encoding a protein having 70% or more, specifically 80% or more, more specifically 90% or more, much more specifically 95% or more homology thereto, much more specifically 98% or more, and most specifically 99% or more homology to the sequence, and may especially include a nucleotide sequence of SEQ ID NO: 1 or 3.
  • a polynucleotide encoding NCgl2523 of the present invention may be hybridized in stringent conditions with a probe of a nucleotide sequence represented by SEQ ID NO: 1 or 3, or a probe derived from the nucleotide sequence, and may be a variant encoding functionally normal NCgl2523.
  • stringent conditions refers to conditions which enable a specific hybridization between polynucleotides. For example, such stringent conditions are described in detail in literature (i.e., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 ; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York ).
  • the present invention may provide a recombinant microorganism showing putrescine production at a high yield by inhibiting NCgl2522 expression resulting from inactivating NCgl2523 activity.
  • putrescine productivity may be enhanced in a microorganism having amino acids having sequences similar to that of NCgl2523, in which such amino acids consist of NCgl2522 and an operon, or a microorganism in which NCgl2523 acts similarly as an NCgl2522 expression modulator.
  • activation refers to not expressing a gene encoding a corresponding polypeptide, showing a certain reduction in gene expression, not producing a corresponding functional polypeptide, even when expressed.
  • inactivation refers not only to completely inactivating a gene encoding a corresponding polypeptide, but also to a weakened or significantly reduced expression compared to the wild-type, thereby practically not expressing the gene. Therefore, gene inactivation may be complete (knockout) or partial (for example, a hypomorph which shows gene expression below the normal level, or a product of a mutant gene which shows partial reduction in activity in effect of a hymorph).
  • inactivation of NCgl2523 may be induced by:
  • the term "vector” refers to a DNA construct including a nucleotide sequence encoding a desired protein, which is operably linked to an appropriate expression regulatory sequence to express the desired protein in a suitable host cell.
  • the regulatory sequence includes a promoter that can initiate transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome-binding site, and a sequence regulating the termination of transcription and translation.
  • the vector used in the present invention is not particularly limited, as long as it is able to replicate in host cells, and any vector known in the art may be used.
  • Examples of conventional vectors may include a natural or recombinant plasmid, cosmid, virus, and bacteriophage.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as a phage vector or cosmid vector
  • pBR types, pUC types, pBluescriptII types, pGEM types, pTZ types, pCL types, pET types, etc. may be used as a plasmid vector.
  • a vector usable in the present invention is not particularly limited, and any known expression vector may be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, or pCC1BAC vector may be used.
  • polynucleotide encoding a desired protein in chromosomes may be replaced by a mutated polynucleotide using a vector for chromosomal insertion.
  • the insertion of the polynucleotide into the chromosome may be performed by any method known in the art such as homologous recombination, without being particularly limited thereto.
  • the term "transformation” refers to introduction of vectors including a polynucleotide encoding target proteins into host cells so that proteins encoded by the polynucleotide are expressed in host cells.
  • the transformed polynucleotide includes all whether it is integrated into chromosomes of host cells or exists extrachromosomally.
  • the polynucleotide includes DNA and RNA encoding target proteins.
  • the polynucleotide may be introduced in any form, as long as it can be introduced into host cells and expressed therein.
  • the polynucleotide may be introduced into host cells in the form of an expression cassette which is a gene construct including all elements required for its autonomous expression.
  • the expression cassette includes a promoter operably linked to the polynucleotide, transcriptional termination signals, ribosome-binding sites, or translation termination signals.
  • the expression cassette may be in the form of a self-replicable expression vector.
  • the polynucleotide may be introduced into host cells as it is and operably linked to sequences required for expression in host cells, without being limited thereto.
  • operably linked refers to a functional linkage between a polynucleotide sequence encoding desired proteins and a promoter sequence which initiates and mediates transcription of the polynucleotide sequence.
  • 2) modification of a regulatory sequence to decrease an expression of the polynucleotide may be performed by inducing modification in a regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in a nucleotide sequence or replacing with a nucleotide with a weaker activity, in order to weaken the activity of the regulatory sequence, without being particularly limited thereto.
  • the regulatory sequence may include a promoter, an operator sequence, a sequence encoding ribosome-binding site, and a sequence regulating termination of transcription and translation, without being limited thereto.
  • modification of the polynucleotide sequence on chromosome may be performed by inducing modification in a regulatory sequence deletion, insertion, non-conservative or conservative substitution, or a combination thereof in a nucleotide sequence, or replacing with a nucleotide with a weaker activity, in order to weaken the protein activity, without being limited thereto,
  • a microorganism having putrescine productivity or "a microorganism producing putrescine” refers to a microorganism naturally having putrescine productivity or a microorganism, in which putrescine productivity is incorporated into a parent strain having no putrescine productivity.
  • the microorganism producing putrescine may be, without being particularly limited thereto, a microorganism having improved productivity of ornithine to be used as a raw material for putrescine biosynthesis, in which the microorganism is modified to have higher activities of acetylglutamate synthase converting glutamate to acetylglutamate (N-acetylglutamate) or ornithine acetyltransferase (ArgJ) converting acetyl ornithine to ornithine, acetylglutamate kinase (ArgB) converting acetyl glutamate to acetylglutamyl phosphate (N-acetylglutamyl phosphate), acetyl-gamma-glutamyl phosphate reductase (ArgC) converting acetyl glutamyl phosphate to acetyl glutamate semialdehyde (N-
  • the microorganism is modified to inactivate endogenous activity of ornithine carbamoyltransfrase (ArgF) involved in arginine synthesis from ornithine, glutamate exporter (NCgl1221), and/or acetyltransferase which acetylizes putrescine and/or is modified to introduce activity of ornithine decarboxylase (ODC).
  • ArgF ornithine carbamoyltransfrase
  • NRC glutamate exporter
  • ODC ornithine decarboxylase
  • the ornithine carbamoyltransfrase(ArgF), glutamate exporter (NCgl1221), ornithine decarboxylase(ODC), acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetyl glutamate synthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD), may include specifically an amino acid sequence each represented by SEQ ID NO: 8, 9, 10, 11, 12, 13, and 14 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more homology to the sequence, without being particularly limited thereto.
  • an acetyltransferase which acetylizes putrescine, may include specifically an amino acid sequence represented by SEQ ID NO: 15 or 16 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more homology to the sequence, without being particularly limited thereto.
  • an increase in activity in the present invention may be carried out by:
  • 2) modification of a regulatory sequence to increase the polynucleotide expression may be performed by inducing modification in a sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing with a nucleotide sequence with enhanced activity, in order to enhance the activity the regulatory sequence, without being particularly limited thereto.
  • the regulatory sequence may include a promoter, an operator sequence, a sequence encoding a ribosome-binding site, a sequence regulating termination of transcription and translation, etc., without being particularly limited thereto.
  • a strong heterologous promoter may be linked instead of the original promoter.
  • the strong promoter are CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, etc., and more specifically, may be operably linked to a Corynebacterium -originated promoter, lysCP1 promoter ( WO2009/096689 ), or CJ7 promoter ( KR Patent No. 0620092 and WO2006/065095 ) and increase an expression of a polynucleotide encoding the enzyme, but are not limited thereto.
  • 3) modification of a polynucleotide sequence on chromosome may be performed by inducing modification in a regulatory sequence by deletion, insertion, non-conservative or conservative substitution or a combination there of in a nucleotide sequence, or by replacing with a polynucleotide sequence which is modified to have enhanced activity, in order to enhance activity of the polynucleotide sequence, without being particularly limited thereto.
  • inactivation of ornithine carbamoyltransfrase (ArgF), glutamate exporter, and acetyl transferase may be performed by methods of inactivating NCgl2523 as previously mentioned:
  • a microorganism of the present invention is a microorganism having putrescine productivity and includes prokaryotic microorganisms expressing proteins including an amino acid represented by SEQ ID NO: 2.
  • examples of such are Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp., Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio sp., Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp. microorganisms, etc.
  • a microorganism of the present invention may be a microorganism of genus Coryne
  • the present invention uses strains having high-concentration putrescine productivity due to an enhanced putrescine biosynthesis pathway from glutamate, which are microorganisms of genus Corynebacterium deposited under Accession Nos. KCCM11138P ( KR Patent No. 2012-0064046 ) and KCCM11240P ( KR Patent No. 2012-0003634 ).
  • the present invention uses KCCM11138P and KCCM11240P, which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13032, and DAB12-a ( KR Patent No. 2013-0082478 ) and DAB12-b ( KR No. 2013-0082478 ; DAB12-a ⁇ NCgl1469), which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13869, each having identical genotypes.
  • ATCC13869 strain may be obtained from American Type Culture Collection (ATCC).
  • the present inventors named a microorganism of genus Corynebacterium having enhanced putrescine productivity due to inactivation of NCg12523 activity in Corynebacterium glutamicum KCCM11240P, which is a putrescine-producing strain, as Corynebacterium glutamicum CC01-0844, and deposited it to Korean Culture Center of Microorganisms (KCCM) under Budapest Treaty as Accession No. KCCM11520P on February 25, 2014.
  • KCCM Korean Culture Center of Microorganisms
  • the present invention provides a method of producing putrescine including:
  • culturing the microorganism may be performed by known batch culturing methods, continuous culturing methods, fed-batch culturing methods, etc., without being particularly limited thereto.
  • culture conditions may be maintained at optimal pH (e.g., pH 5 to 9, specifically pH 6 to 8, and most specifically pH 6.8) using basic compounds (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid), and at an aerobic condition by oxygen or oxygen-containing gas mixture to a cell culture, without being particularly limited thereto.
  • the culture temperature may be maintained at 20°C to 45°C and specifically at 25°C to 40°C, and cultured for about 10 to 160 hours. Putrescine produced by the cultivation may be exported to the culture medium or remain in cells.
  • the used culture medium may include sugar and carbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose, molasse, starch, and cellulose), oil and fat (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acid (e.g., palmitic acid, stearic acid, and linoleic acid), alcohol (e.g., glycerol and ethanol), and organic acid (e.g., acetic acid) individually or in combination as a carbon source; nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn solution, soybean meal powder, and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) individually or in combination as a nitrogen source; potassium dihydrogen phosphate, dipotassium phosphate, or sodium
  • Separating putrescine produced in the culturing step of the present invention may be performed using a suitable method known in the art depending on culturing methods such as batch, continuous or fed-batch culturing methods, etc., thereby collecting desired amino acids from the culture.
  • a microorganism of genus Corynebacterium having enhanced putrescine productivity of the present invention is modified to inactivate activity of a protein including an amino acid sequence of SEQ ID NO: 2, thereby inducing enhanced activity of a protein which is expected to be a putrescine exporter, specifically NCgl2522, and increasing putrescine export to the outside of cells, and thus may effectively produce putrescine.
  • Fig. 1 is a schematic diagram showing combined structures of NCgl2523 and the adjacent.
  • Fig. 1 is a schematic diagram showing a combined structure of NCgl2522-NCgl2523-NCgl2524 which is a combined structure of adjacent genes of a microorganism including NCgl2523 (Type 1); a structure of combined NCgl2522-NCgl2523 and individual NCgl2524 (Type 2); or a combined structure of NCgl2522-NCgl2523 (Type 3).
  • NCgl2523 derived from Corynebacterium glutamicum ATCC13032 has an effect in putrescine productivity.
  • a pair of primers of SEQ ID NOs: 4 and 5 for obtaining homologous recombination fragments at the N-terminal region of NCgl2523, and a pair of primers of SEQ ID NOs: 6 and 7 for obtaining homologous recombination fragments at the C-terminal region of NCgl2523 were constructed as shown in Table 1.
  • NCgl2523-del-F1_BamHI (SEQ ID NO: 4) CGGGATCCATGACTACCTCGCAGCGTTTC NCgl2523-del-R1_SalI (SEQ ID NO: 5) ACGCGTCGACCTAGTGCGCATTATTGGCTCC NCgl2523-del-F2_SalI (SEQ ID NO: 6) ACGCGTCGACAGCCATGCTGGAAACAATTCTCG NCgl2523-del-R2_XbaI (SEQ ID NO: 7) CTAGTCTAGAGAGAGCTGCGCATAGTACTG
  • PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and two pairs of primers, thereby amplifying PCR fragments at the N-terminal and C-terminal regions of NCg12523 gene. Desired fragments were obtained via electrophoresis of the PCR fragments. Here, PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, and 30 seconds of extension at 72°C. The thus obtained fragments of the N-terminal and C-terminal regions were treated with restriction enzymes BamH I and Sal I, and restriction enzymes Sal I and Xba I, respectively. The treated fragments were cloned into pDZ vectors treated with restriction enzymes BamH I and Xba I, thereby constructing pDZ-1'NCgl2523(K/O)plasmids.
  • the pDZ-1'NCgl2523 (K/O) plasmids were each transformed into KCCM11138P ( KR Patent No. 10-1348461 ) and KCCM11240P ( KR Patent No. 2013-0082478 ) by electroporation to obtain transformants.
  • the transformants were plated and cultured on BHIS plate media (Braine heart infusion (37 g/L), sorbitol (91 g/L), and agar (2%)) containing kanamycin (25 ⁇ g/mL) and X-gal(5-bromo-4-chloro-3-indolin--D-galactoside). From the formed colonies, blue colonies were selected as strains introduced with pDZ-1'NCgl2523(K/O) plasmids.
  • the selected strains is cultured in a CM medium (glucose 10 g/L, polypeptone (10 g/L), yeast extract (5 g/L), beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), and pH 6.8) at 30°C for 8 hours.
  • a CM medium glucose 10 g/L, polypeptone (10 g/L), yeast extract (5 g/L), beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), and pH 6.8
  • the diluted samples were plated and cultured in an X-gal-containing solid medium to form colonies. From the formed colonies, white colonies, which appeared at a relatively low frequency, were finally selected as strains with deletion of a gene encoding NCgl2523 by a secondary crossover.
  • PCR was performed using a pair of primers of SEQ ID NOs: 4 and 7 to confirm deletion of a gene encoding NCgl2523 in the finally selected strains.
  • the Corynebacterium glutamicum mutants were each named as KCCM11138P ⁇ NCgl2523 and KCCM11240P ⁇ NCgl2523.
  • NCgl2523-deleted strains were constructed from DAB12-a ( KR Patent No. 2013-0082478 ) and DAB12-b ( KR Patent No. 2013-0082478 ; DAB12-a ⁇ NCgl1469), which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13869.
  • PCR was performed using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and a pair of primers of SEQ ID NOs: 4 and 7.
  • PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, and 1 minute 30 seconds of extension at 72°C
  • a gene encoding NCgl2523 derived from Corynebacterium glutamicum ATCC13869 was found to include a nucleotide sequence represented by SEQ ID NO: 3. Further, an amino acid sequence of proteins encoded by the gene was compared to an amino acid sequence of NCgl2523 (SEQ ID NO: 2) derived from Corynebacterium glutamicum ATCC13032, and the result showed 100% homology.
  • PCR was performed using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and two pairs of primers described in Table 1 as in Example ⁇ 1-1>, and PCR fragments of the N-terminal C-terminal regions of NCgl2523 gene were amplified, thereby obtaining desired fragments via electrophoresis.
  • PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, and 30 seconds of extension at 72°C.
  • the obtained fragments of the N-terminal and C-terminal regions were treated with restriction enzymes BamH I and Sal I, and restriction enzymes Sal I and Xba I, respectively.
  • the treated fragments were cloned into pDZ vectors treated with restriction enzymes BamH I and Xba I, thereby constructing pDZ-2'NCgl2523(K/O) plasmids.
  • ⁇ 1-1> and ⁇ 1-2> were compared for putrescine productivity.
  • Corynebacterium glutamicum mutants (KCCM11138P ⁇ NCgl2523, KCCM11240P ⁇ NCgl2523, DAB12-a ⁇ NCgl2523, and DAB12-b ⁇ NCgl2523) and 4 different types of parent strains (KCCM11138P, KCCM11240P, DAB12-a, and DAB12-b) were each plated on 1 mM arginine-containing CM plate media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 ⁇ L of 50% NaOH, and 2% agar, at pH 6.8, base on 1 L) and cultured at 30°C for 24 hours.
  • arginine-containing CM plate media 1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 ⁇ L of 50% NaOH, and 2% agar, at pH
  • intercellular putrescine concentrations were measured in Corynebacterium glutamicum mutant KCCM11240P ⁇ NCgl2523 and parent strain KCCM11240P by extraction using an organic solvent.
  • Intracellular metabolite analysis was carried out in accordance with a method described in literature ( Nakamura J et al., Appl. Environ. Microbiol., 73(14): 4491-4498, 2007 ).
  • NCgl2523 is deleted in Corynebacterium glutamicum mutant KCCM11240P, inhibition of NCgl2522 expression is withdrawn and NCgl2522 activity is enhanced, thereby enhancing putrescine-exporting ability and subsequently exporting putrescine produced inside the cell to the outside of cells efficiently.
  • NCgl2523 which is derived from Corynebacterium glutamicum ATCC13032, using KEGG, MetaCyc Database and NCBI blastP.
  • NCgl2523 derived from Corynebacterium glutamicum ATCC13032
  • KEGG KEGG
  • MetaCyc Database NCBI blastP.
  • NCgl2523 a combined structure of NCgl2523, and NCgl2522 and NCgl2524, which constitute an operon in the genome of each microorganism was examined.
  • NCgl2522 is cgmA, which is a major facilitator superfamily permease belonging to DHA2 family
  • gene name of NCgl2523 is cgmR, which is TetR-family transcriptional regulator.
  • NCgl2524 has not been attributed with a function, but is a major facilitator superfamily permease belonging to UMF1 family.
  • NCgl2524 may be present in the same operons (Type 1), present in a different position in the genome (Type 2), or not present in the genome (Type 3), depending on microorganisms.
  • each of analyzed microorganisms was classified into 3 types according to a combined structure of genes adjacent to NCgl2523.
  • microorganisms which are expected to have NCg12523 were classified into a combined structure of NCgl2522-NCgl2523-NCgl2524 (Type 1); a structure of combined NCgl2522-NCgl2523 and individual NCgl2524 (Type 2); or a combined structure of NCgl2522-NCgl2523 (Type 3) ( Fig. 1 ).
  • the classification results are shown in Table 4.
  • Pectobacterium carotovorum subsp. carotovorum PCC21 Promicromonospora sukumoe, Propionibacterium acidipropionici, Propionibacterium freudenreichii, Proteobacteria, Providencia stuartii ATCC 33672, Pseudomonas aeruginosa, Pseudomonas cremoricolorata, Pseudomonas geniculata, Pseudomonas stutzeri, pseudonocardia dioxanivorans, Renibacterium salmoninarum, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus jostii, Rhodococcus opacus B4, Rhodococcus opacus PD630, Rhodococcus pyridinivorans, Saccharomonospora viridis, Saccharopolyspora erythraea, Salinispora
  • NCgl2522-NCgl 2523-NCgl2524 Corynebacterium callunae, Corynebacterium casei, Corynebacterium glutamicum AR1, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum ATCC 21831, Corynebacterium glutamicum K051, Corynebacterium glutamicum MB001, Corynebacterium glutamicum R, Corynebacterium glutamicum SCgG1, Corynebacterium glutamicum SCgG2, Corynebacterium vitaeruminis 2 Structure of combined NCgl2522-NCgl 2523; individual NCgl2524 Actinoplanes missouriensis, Acti
  • carotovorum PCC21 Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas cremoricolorata, Pseudomonas stutzeri, Sanguibacter keddieii, Serratia liquefaciens, Serratia marcescens, Serratia plymuthica, Serratia proteamaculans, Serratia sp., Sodalis sp. HS1, Stenotrophomonas maltophilia, Xenorhabdus nematophila, Yersinia enterocolitica
  • putrescine productivity may be enhanced as in the present invention by inactivating genes encoding NCgl2523 and an amino acid sequence similar thereto, for microorganisms having an amino acid including a sequence similar to NCgl2523 and consisting of an operon with a protein expected to be a putrescine exporter, such as NCgl2522, as in the classification.

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